The objective of this grant application is to develop a new method to better characterize Epstein-Barr virus (EBV)-infected cells from aged subjects. EBV is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We have new data which shows that aged subjects have plasma viremia (EBV DNA). In addition, molecular analysis revealed frequent transcription of early and late structural genes in EBV-infected B-cells. Thus, we have an immediate need to develop assays to better characterize EBV-infected cells. We have encouraging preliminary data that demonstrates our ability to detect EBV-infected B cells at the single-cell level using fluorescent in situ hybridization (FISH) combined with flow cytometry. This application is not hypothesis driven per se but supports the development of a research technology that will be used to characterize EBV-infected B-cells in archived specimens from aged subjects that have high viral load as determined by PCR. The specific aims are: (1) Design and test multiple antisense probes (MAPs) that target EBERs;and (2) Application of MAPs to characterize EBV-infected B-cells from aged subjects. The successful completion of this project will yield a novel method to detect EBV-infected B cells in peripheral blood, and this methodology will be incorporated into a more comprehensive R01 application to investigate the immunobiology of EBV in aging. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is an important pathogen and results in considerable cost to the health care system. EBV is the causative agent of infectious mononucleosis as well as nasopharyngeal carcinoma, Burkitt's lymphoma, and other B-cell lymphomas in immunosuppressed individuals. Although much has been learned about EBV from studies of in vitro transformed B-lymphoblastoid cell lines, critical gaps in knowledge regarding EBV pathogenesis in vivo remain to be answered, including how EBV persists in spite of the continuous immune surveillance. We will develop a novel FISH-based assay to detect EBV-infected cells in peripheral blood at the single-cell level which can be used to better understand the immunology of EBV in aging.